JP2009247355A - Analytical processing and detection device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an analytical processing and detection device, enabling the detection of detecting signals in the fluid of a reaction container under the presence of magnetic particles. <P>SOLUTION: This analytical processing and detection device is provided by having a rack capable of holding at least one reaction container and a ≥1 magnetic unit for exerting a magnetic field on at least the one reaction container containing the magnetic particles, wherein the ≥1 unit is linked with the rack reversibly or irreversibly, and the magnetic field is characterized by comprising a unit sequestering the magnetic particles at the inside wall of the at least the one reaction container and a detection unit for detecting the signals of the fluid in the reaction container. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to an analysis process and a detection apparatus that enable detection signals in a fluid in a reaction vessel to be detected in the presence of magnetic particles.

  In general, for an analysis method based on gene or protein expression or genetic analysis, an analyte in a biological sample is generated by adsorption (binding) to a solid phase such as magnetic particles, and then a detection signal is obtained. In order to obtain an analytical result that can be used further in this way and thus can be used in monitoring for diagnosis or treatment of disease. In the field of PCR diagnostics, the analyte bound to the particles is washed and eluted prior to further processing. The eluate is then transferred to a new container for PCR amplification and detection of the amplified PCR product in the liquid. In such a setting, without amplification of the particles, PCR amplification and detection is performed in the absence of particles, which will disturb the optical detection of the detection signal in the liquid. However, elution and transfer of the specimen has a disadvantage that the specimen contained in one reaction vessel cannot be completely transferred due to residual liquid remaining in the reaction vessel (interstitial volume). In the field of PCR diagnostics where a small amount of sample is processed and analyzed, such incomplete transfer of liquids degrades analytical sensitivity.

  WO 03/057910 discloses a method for amplifying and optically detecting a detection signal in a liquid phase of a container without removing magnetic particles. The particles can be fixed to the bottom of the container by sedimentation.

  The present invention provides a rack for receiving at least one reaction vessel, the rack including at least one gap capable of receiving the reaction vessel, and at least a magnetic particle capable of binding to an analyte contained in a fluid. One or more magnetic units for exerting a magnetic field on one reaction vessel, wherein the one or more units are coupled to the rack and by a magnetic field generated by the at least one magnetic unit, The magnetic particle includes a unit sealed on the inner wall of the at least one reaction vessel and a detection unit for detecting a signal of a fluid in the reaction vessel, in order to detect a signal from the reaction vessel. In addition, the detection unit is disposed in the apparatus, and the reaction vessel containing the magnetic particles includes a detection unit held in the rack. The present invention relates to an analysis process and a detection device, wherein a rack is provided in the apparatus for detecting a signal from the reaction vessel while holding a reaction vessel containing the magnetic particles in the rack. It is.

  Further, the apparatus described below is used in a method of processing the contents in at least one reaction vessel and quantifying the amount of analyte bound to magnetic particles present in each of the reaction vessels. In addition, in this method, one or more magnetic units can be brought into contact with the rack to accurately and highly sensitively process and quantify the specimen in the presence of magnetic particles. The magnetic unit is connected to the rack so that the magnetic particles are sealed on the side wall of the reaction vessel. By sealing the magnetic particles to the side walls, the liquid in the well is essentially free from magnetic particles that interfere with the detection method. The center of the well is preferably free of magnetic particles. The term “coupled” as used herein relates to any type of contact between the magnet and the rack. Conversely, the magnet may be coupled to the rack. It is preferable that the magnets are stably integrated in the rack.

  In this way, the detection signal is weakened only minimally by the magnetic particles, so that the specimen can be processed without the need to separate the eluate from the magnetic particles (FIG. 3). The term “separate the eluate from the magnetic particles” is understood to mean that the eluate is separated and that after separation there is no longer any physical contact with the magnetic particles. FIG. 1 shows how the detection signal is weakened by the presence of unblocked or poorly blocked magnetic particles. Due to the magnetic sealing of the magnetic particles in the container, material loss due to the interstitial volume during pipetting of the eluate can be avoided. Furthermore, although magnetic particles are not removed, blockage to the side walls of the reaction vessel reduces the reduction in signal to noise ratio caused by suspended or settled particles and thus improves detection sensitivity.

  The term “reaction vessel” as used herein relates to a vessel that can hold a liquid and in which a reaction such as a chemical reaction or an enzymatic reaction takes place. The reaction vessel comprises an opening at the top, a side wall, and a bottom. The opening may be sealed with a cap or foil. In a preferred embodiment, a microtiter plate comprising a plurality of reaction vessels is employed, preferably a 96 well microtiter plate. The shape of the microtiter plate is generally known. The microtiter plate is preferably white. The white plate improves the efficiency of fluorescence excitation and detection by allowing multiple light reflections in the beam. The white plate is typically a polypropylene plate containing titanium dioxide as a whitening agent. It is important that the plate lacks trace amounts of material that contributes to plate autofluorescence. In a preferred embodiment, the shape of the reaction vessel is determined by both thermal considerations and liquid handling considerations.

  From a thermal point of view, shapes that are ideally high and thin cylinders or very short and flat cylinders maximize the direct thermal contact with the heat block in order to increase the temperature gradient and thermal equilibrium. It should be something like The vessel wall must have a sufficient angle of inclination from the vertical so that the microtiter plate can be easily removed from the thermal circulator after PCR amplification. Furthermore, the shape of the container must be sufficiently wide and must have a round bottom to avoid foam formation during liquid dispensing.

  The term “magnetic particle” relates to a particle comprising a magnetic material capable of binding an analyte in a fluid. The specimen is preferably bound directly to the magnetic particles. The term includes magnetic particles with or without a silica surface. The magnetic particles are preferably magnetic glass particles.

In a preferred embodiment, if the analyte is a nucleic acid, the magnetic glass particles are magnetic glass particles comprising an unmodified silica surface. A preferred embodiment of the magnetic glass particles is disclosed in WO 96/41811. The magnetic glass particles are a solid dispersion in a glass having a small magnetic core, that is, a glass droplet in which a very small magnetic material is dispersed. These objects that are made magnetic are attracted to a magnet, ie, for example, a ferrimagnetic or ferromagnetic material or a superparamagnetic material. Paramagnetic materials are not useful because they are only drawn very weakly by magnets that are not sufficient for the method according to the invention. Ferrimagnetic materials or ferromagnetic materials are preferred, especially when the material is not pre-magnetized. Preliminary magnetization in this context is understood to mean contacting with a magnet that increases remanence. A preferred magnetic material is iron or iron oxide such as, for example, magnetite (Fe ₃ O ₄ ) or Fe ₂ O ₃ , preferably gamma Fe ₂ O ₃ . In principle, barium ferrite, nickel, cobalt, Al-Ni-Fe-Co alloys, or other ferrimagnetic or ferromagnetic materials can be used. The magnetic glass particles described in WO 96/41811 pamphlet, WO 00/32762 pamphlet or WO 98/12717 pamphlet are particularly preferred according to the present invention.

  In a highly preferred embodiment of the invention, the magnetic glass particles having an unmodified glass surface are used when the use of magnetic glass particles as iron is an inhibitor of subsequent amplification reactions, i.e. when iron is an enzyme inhibitor. It has a low iron filter which is essential for the present invention. This is therefore an important property of magnetic glass particles with an unmodified glass surface. In the most preferred embodiment of the present invention, the magnetic glass particles having an unmodified glass surface are those described in EP 2000110165.8, which are MagNA pure LCDNA isolations. Available in Kit I (Roche, Mannheim, Germany). Their production is summarized below.

The most preferred MGP according to the present invention, which is also provided in MagNA pure LC DNA isolation kit I (Roche, Mannheim, Germany), is manufactured according to EP 1154443. They are manufactured from CERAC, which consists of a magnetic substance or magnetic pigment having a diameter of about 23 nm (γ-Fe ₂ O _3. CERAC: PO Box 1178, Milwaukee, Wisconsin, USA 53201-1178; It is also produced by the sol-gel method described in EP 1544443 using article number I-2012).

  According to the present invention, an “analyte” is understood to be a molecule or a collection of molecules including cellular components of cells or viruses found in a sample. Thus, as a non-limiting example, an analyte may be an important nucleic acid or important protein to be considered, and its presence or absence may indicate a human or animal condition or disease, so its presence Alternatively, the absence or concentration in the biological sample is determined. In addition, fragments of such molecules found in the sample are included within the scope of the term “analyte”. In one preferred embodiment, the sample is a biological sample, more preferably a nucleic acid. The nucleic acid may be RNA or DNA or a derivative thereof. In a more preferred example, the specimen is a virus, more preferably hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), human papilloma virus (HPV). Parvovirus B19, CT / NG. In a more preferred example, the specimen may be a bacterium such as MAI or MTB.

  The term “fluid” refers to any type of liquid that contains magnetic particles capable of binding an analyte. This may be a biological sample as described below or a buffer solution. In one embodiment, the buffer solution is an elution buffer. The low salinity elution buffer is in particular a buffer with a content of less than 0.2 mol / l. In a particularly preferred embodiment, the elution buffer comprises a Tris material for buffering purposes, particularly a Tris buffer having a pH of about 7 or higher. In another particular embodiment, the elution buffer is desalted water.

As used herein, the term “rack” refers to a unit having a gap that can hold at least one reaction vessel. In a preferred embodiment, the rack is a thermally conductive rack. This means that the rack material preferably has the necessary high thermal diffusivity for fast thermal ramps. This is preferably a high thermal conductivity of 200 W / (mK) or more, more preferably at least 221 W / (mK), preferably less than 1000 J / (kgK), more preferably less than or equal to 900 J / (kgK). And a low density of preferably less than 12 kg / dm ³ , more preferably less than or equal to 10.5 kg / dm ³ . In a more preferred embodiment, the rack material described above consists of aluminum or silver. The thermal conductivity of silver is 429 W / (mK) and that of aluminum is 221 W / (mK). The heat capacity of silver is 232 J / (kgK) and that of aluminum is 900 J / (kgK). The density of silver is 10.5 kg / dm ³ and that of aluminum is 2.7 kg / dm ³ . In the most preferred embodiment, the rack described above is a heating block in a thermal cycler. The heating block is controlled to change between at least two temperatures in one cycle, preferably between annealing temperature, incubation temperature and denaturation temperature. The temperature of the heating block can be changed rapidly. Cooling may be accomplished by bringing the heating block into contact with a heat sink, such as a liquid. Alternatively, it may be achieved by a thermoelectric element such as a Peltier element. A combination of a resistance heater and a thermoelectric cooling element, or a thermoelectric element used for both heating and cooling is an equipment configuration that is most frequently used apart from heating and cooling using air.

  The term “magnetic unit” as used herein relates to a device capable of exhibiting a magnetic field. The magnetic unit may be an electromagnetic unit or a permanent magnet.

  In one embodiment, the magnetic field shown on the reaction vessel containing the magnetic particles described above can be increased or decreased. Increasing the magnetic field may be achieved by moving the magnet in close proximity to the rack holding the reaction vessel. Thus, reduction may be achieved by removing magnets from the rack.

  In the embodiment of the invention described above, one or more units for exerting a magnetic field on at least one reaction vessel containing magnetic particles in a fluid are stably integrated in the rack. One or more permanent magnets. In a preferred embodiment, the one or more magnetic units are thermally stable at temperatures up to 140 ° C. This means that the ability of a permanent magnet to generate a magnetic field is not weakened by such high temperatures. This allows the generation of a magnetic field even during thermal cycling, which requires heating the liquid contents in the reaction vessel to a temperature as high as 100 ° C. The heat block itself can be temporarily heated to a temperature of 110 ° C. This so-called block overshoot is used to accelerate the liquid equilibrium to the final temperature. In a more preferred embodiment, the one or more permanent magnets are thermally stable up to a temperature of 180 ° C. In an even more preferred embodiment, the one or more permanent magnets are thermally stable up to a temperature of 250 ° C. In the most preferred embodiment, the one or more permanent magnets are comprised of an SmCo alloy.

  As described above, the shape and arrangement of the permanent magnets described above also affect the sealing efficiency of the magnetic particles on the inner wall of the reaction vessel. Thus, in a preferred embodiment, the gap for receiving the reaction vessel is arranged between at least two permanent magnets so that the isomagnetic pole of the permanent magnet faces the same gap.

  In one more preferred embodiment of the apparatus described above, the two or more permanent magnets are pin-shaped and the magnets extend over the entire width or length of the rack. In another more preferred embodiment, the permanent magnet is pin-shaped, where the length of the magnet substantially corresponds to the diameter of the gap in the rack, where the magnet is equal to the permanent magnet, etc. The magnetic poles are arranged between two gaps for receiving the rack reaction vessels so that they face the same gap. In a more preferred embodiment, the magnetic field is adjusted to project to a horizontal plane and minimize the area of the collected beads. Most preferably, the beads are well concentrated just below the liquid surface.

  The apparatus described above is suitable for detecting signals that are weakened by the presence of magnetic particles. Thus, the term “detection unit” relates to a detection unit that can detect signals that are weakened by the presence of magnetic particles. Preferably, the detection unit is an optical unit. More preferably, the signal is fluorescence. The detection unit may comprise a photodiode or a CCD chip (for example as described in WO 99/60381 or DE 19748211). The detection unit further comprises a light source for excitation light fermentation such as a 100 watt halogen lamp (WO 99/60381) or an LED array. Further, the detection apparatus may include a beam splitter (German Patent No. 10131687, German Patent No. 10155142), if necessary. Furthermore, the detection device may comprise a Fresnel lens (US Pat. No. 6,246,525), a field lens (EP 1681556) or one or more telecentric lenses (US Pat. No. 6,498,690). Good.

  In contrast to the apparatus described above, the present invention also relates to an analysis system including the apparatus described above.

Such an analysis system and apparatus is a method for quantifying the amount of analyte bound to magnetic particles present in a sample fluid in a reaction vessel, comprising:
The method
a) providing a biological sample containing the analyte in at least one reaction vessel;
b) binding magnetic particles to the analyte in the at least one reaction vessel;
c) can be used to perform a method comprising the step of processing the analyte bound to magnetic particles in the at least one reaction vessel to detect a detection signal. The detection signal is a signal disturbed by the presence of the magnetic particles. In a preferred embodiment, the detection signal is an optical signal. Furthermore, in step c), at least one reaction vessel is placed in the analytical processing unit.

  In step d), suspended matter is generated by using a magnetic field for the contents of the at least one reaction vessel. The magnetic particles are sealed on the inner wall of the reaction vessel by the magnetic field. Step d) is followed by step e) in which a detection signal corresponding to the analyte concentration in the suspension of the reaction vessel is detected, preferably by optical detection, in the presence of the blocked magnetic particles. . In the method of the present invention, the steps c) to e) are performed in the same analytical processing unit.

  All processes are preferably automated. Automated methods are performed using devices or machines that can be automated without any human control or influence on the steps of the automatable method.

  In a preferred embodiment of the present invention, the method is performed in a high-speed mass processing format, i.e. the automated method allows the method and the machine or apparatus used to be used for high-speed mass processing of more than 100 samples in a short time. It is done in a high-speed, high-volume processing format that means it is optimized.

  The term “floating matter” as used herein relates to the liquid in the reaction vessel after the magnetic particles have been blocked by a magnetic field. In this way, the suspended matter refers to the liquid in the reaction vessel after the magnetic particles are sealed by the magnetic field, and the magnetic particles form a pellet at the location of the side wall of the reaction vessel to be sealed.

  The term “biological sample” as used herein relates to a sample derived from a biological organism. In embodiments of the invention, not only abbreviated cells from multicellular organisms, but also viruses, bacterial cells, eg human and animal cells such as leukocytes, and haptens, antigens, antibodies and nucleic acids, plasma, brain Includes spinal fluid, saliva, biopsy specimens, bone marrow, oral lavage fluid, immunologically active low molecular compounds and high molecular compounds such as serum, tissue, urine or mixtures thereof. Thus, the biological sample may be solid or fluid. In a preferred embodiment of the invention, the biological sample is a fluid from a human or animal torso. A biological sample that is a fluid is also called a sample fluid. The biological sample is preferably blood, plasma, serum or urine. The plasma is preferably EDTA plasma, heparin plasma, or citrated plasma. In an embodiment of the present invention, the biological sample comprises bacterial cells, eukaryotic cells, viruses or mixtures thereof. In a preferred embodiment of the invention, the virus is hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), human papilloma virus (HPV), parvovirus. B19, CT / NG, CMV, and the bacteria are MTB or MAI. The biological sample may be of the type used for environmental analysis, food analysis, or for example, bacterial cultures or phage lysates.

  When a biological sample can be used in the method according to the present invention without pretreatment, a biological sample containing a mixture of non-target nucleic acids and biological compounds comprising target nucleic acids need not be lysed. However, the biological sample containing the non-target nucleic acid and the target nucleic acid is lysed to make a mixture of biological compounds containing the non-target nucleic acid and the target nucleic acid. Thus, biological compounds, non-target nucleic acids and target nucleic acids contained in a biological sample are released, thereby creating a mixture of biological compounds comprising non-target nucleic acids and target nucleic acids. Techniques for lysing biological samples are known by experts and may be chemical, enzymatic or physical in nature. Moreover, combinations of these techniques can also be used. For example, lysis can be performed using ultrasound, high pressure, shear, alkali, detergent or chaotropic saline solution, or protease or lipase. For lysis techniques for nucleic acid acquisition, Samlock et al .; “Molecular Cloning, Laboratory Manual Second Addendum”, Cold Spring Harbor Laboratory Press Issue, Cold Spring Harbor, NY, and Ausbell et al .; “Current Protocols in Molecular Biology, 1987”, J. Am. See especially Wiley & Sons, New York.

  The method of the present invention includes the step of binding a specimen to magnetic particles in a reaction vessel.

  The magnetic glass particles used in the present invention will be provided in an essentially different formulation, as described in EP 1544443. It is possible to provide the magnetic glass particles in the form of tablets as a powder or preferably as a suspension. In a preferred embodiment of the invention, these suspensions contain between 5 and 60 mg / ml of magnetic glass particles (MGP). In other embodiments of the present invention, the silica-containing material may contain the chaotropic agent at a concentration of 1-8 mol / l, preferably 2-6 mol / l, more preferably 4-6 mol / l, depending on the situation. It may be suspended in a buffered aqueous solution. Chaotropic salts are sodium iodide, sodium perchlorate, guanidine thiocyanate, guanidine isothiocyanate, and guanidine hydrochloride. The chaotropic agent according to the invention disturbs the regular structure of the aqueous liquid, and if this agent is present in a DNA or RNA-containing solution, the effect that the DNA or RNA binds to the MGP according to the invention. Any chemical substance with Of course, other compounds known to experts in the field are also possible. The purification effect results from the behavior of DNA or RNA that binds to the material with the glass surface under these conditions, ie in the presence of a certain concentration of chaotropic agent, or in acidic conditions. Glass beads with an unmodified glass surface are added to the mixture to bring in a mixture of non-target nucleic acids and biological compounds containing the target nucleic acids and are incubated for a period of time sufficient for binding to occur. . Experts are usually familiar with the duration of the culture process. Incubation times between 10 seconds and 30 minutes may be appropriate for nucleic acids.

  One approach for binding target nucleic acids (also non-target nucleic acids) to magnetic glass particles can be described in detail as follows. Other techniques are known to the skilled worker and have been used successfully. The term “analyte” as used herein relates to a target nucleic acid, but both target and non-target nucleic acids may be bound to magnetic glass particles. In one embodiment, the target nucleic acid is specifically bound to the magnetic particles. This specific binding may be mediated by an oligonucleotide with a sequence complementary to the target sequence, as a non-limiting example, whereby the oligonucleotide is tightly bound to the magnetic glass particle . The binding of the target nucleic acid and / or non-target nucleic acid is preferably achieved in the presence of the above-mentioned concentrations of chaotropic salts.

  Following the binding of the analyte to the magnetic particles, the magnetic particles with the bound analyte are separated from the sample. Depending on the situation, the magnetic particles with the bound analyte may be washed using a wash buffer.

  This is done by separating the material bound to the magnetic particles by applying a magnetic field. For example, the magnetic particles can be pulled toward the wall of the container in which the culture is performed. Thereafter, the liquid containing biological compounds that are not bound to the magnetic particles can be removed. Thus, the method according to the invention can comprise the step of separating the material with bound non-target nucleic acid and / or bound target nucleic acid from unbound biological compounds.

  The removal technique used depends on the type of vessel in which the culture was performed. Suitable steps include pipetting or removing the liquid by aspiration. The material with bound DNA or RNA is then preferably described in a mixture of 70 parts by volume ethanol and 30 parts by volume water (“70% ethanol”) or in WO 99/40098. You may wash at least once in the acid washing solution. A cleaning solution is used that does not release target and non-target nucleic acids from the surface of the material but cleans as much as possible unwanted contaminants. This washing step is preferably performed by culturing magnetic particles having an unmodified silica surface using a bound nucleic acid and a target nucleic acid. The material is preferably resuspended during this step. The contaminated cleaning liquid is preferably removed just as in the bonding process described above. After the final washing step, the material can be easily dried in a vacuum or the fluid can be evaporated. A pretreatment step using acetone may be performed.

  Here, the solution containing the purified specimen is further processed immediately. The term “treatment” as used herein relates to the handling of an analyte by chemical or biochemical handling that leads to the generation of a detection signal.

  In a preferred embodiment of the method described above, the processing of the nucleic acid comprises amplification. Amplification is a well-known method for growing specific sequence copies of RNA or DNA that are then detected and analyzed quantitatively or qualitatively. In a preferred embodiment, the treatment comprises elution of the analyte from magnetic beads into the fluid in the reaction vessel. For the elution that occurs, the material with an unmodified silica surface is resuspended in solution in the absence of chaotropic agents and / or organic solvents, or just a small amount. Alternatively, the suspension can be diluted with a solution in the absence of chaotropic agents and / or organic solvents, or with only a small amount. Buffers of this nature are known from DE 37 24 442 and “Analytical Biochemistry” 175 (1988) 196-201.

  An elution buffer with a low salt content is in particular a buffer with a content of less than 0.2 mol / l. In a particularly preferred embodiment, the elution buffer comprises a Tris material for buffering purposes, particularly a Tris buffer having a pH of about 7 or higher. In another particular embodiment, the elution buffer is desalted water.

  The term “processing unit” relates to a unit capable of processing a sample for detection. Such a processing unit may be a thermal cycler. In a preferred embodiment, the processing unit is the analytical processing and detection device described above. In this way, at least steps c) to e) of the method of the invention can be carried out in the analytical processing and detection device.

  For amplification, all reagents necessary for amplification are added to the solution containing the specimen. Otherwise, a solution containing all the reagents necessary for amplification is added to the suspension of material and target nucleic acid having an unmodified silica surface.

  In a preferred embodiment, the target nucleic acid is amplified using the polymerase chain reaction (PCR). The amplification method includes ligase chain reaction (LCR, U and Wallace; Genomics 4 (1989) 560-569 and Balani; Proc. Natl. Acad. Sci. USA 88 (1991) 189-193); Ligase chain reaction (balani; PCR method and application 1 (1991) 5-16); 20 gap-LCR (WO 90/01069); repair chain reaction (EP 439182) 3SR (Quo et al .; Proc. Natl. Acad. I Sci. USA 86 (1989) 1173-1177); Guateri et al., Proc. Natl. Acad. Sci. USA 87 (1990) 1874-1878; WO 92/0880 and NASBA (US Pat. No. 5,130,238). In addition, strand displacement amplification (SDA), transcription-mediated amplification (TMA), and Q0-amplification (for review, see, for example, Welen and Persin; Rev. Microbiol. 50 (1996) 349-373); Abramson and Meyer; “Current Opinions in Biotechnology”, Volume 4 (1993) 41-47).

  A preferred detection signal for the method described above is an optical detection signal. More preferably, the optical detection signal is fluorescence.

  The target nucleic acid may be detected by measuring the intensity of fluorescence during amplification. This method requires real-time fluorescence monitoring. Particularly preferred methods for simultaneously utilizing amplification and detection by measuring the intensity of fluorescence are described in WO 92/02638 and the corresponding US Pat. No. 5,210,015, US Pat. No. 5,804,375 and US Pat. This is the Taqman method disclosed in the specification of No. 5487972. This method takes advantage of the exonuclease activity of the polymerase to generate a signal. Specifically, the first oligonucleotide and the labeled oligonucleotide such that the hybridization state (where the 3 ′ end of the first oligonucleotide is adjacent to the 5 ′ end of the labeled oligonucleotide). And the same target nucleic acid strand comprising a sequence that complements the region of the target nucleic acid to form a double-stranded mixture between the target nucleic acid annealed to The target nucleic acid is obtained by a process comprising contacting a sample with a labeled oligonucleotide comprising a sequence that complements the second region of, but not the nucleic acid sequence defined by the first oligonucleotide. Detected. Thereafter, under conditions sufficient to allow the 5 'and 3' nucleolytic activity of the polymerase to cleave the annealed labeled oligonucleotide and release the labeled fragment, This mixture is treated with a template-dependent nucleic acid polymerase having 5 ′ and 3 ′ nucleolytic activity. The signal generated by hydrolysis of the labeled oligonucleotide is detected and / or measured. Taqman technology eliminates the need to form and detect solid-phase bound reaction complexes. In more general terms, the amplification and / or detection reaction according to the present invention is a homogeneous solution phase assay. A more preferred method is in the LightCycler ™ format (see, eg, US Pat. No. 6,174,670).

  The detection method may include, but is not limited to, binding and insertion of a specific dye, such as ethidium bromide, that inserts into double helix DNA and subsequently changes fluorescence. Excitation light from the light source is directed onto the liquid in the reaction vessel. Fluorescence emission from the liquid following excitation suggests a positive signal and is detected and quantified.

  In an embodiment different from the embodiment in which the analyte is a nucleic acid, the analyte is a polypeptide. In a preferred embodiment, the treatment of the polypeptide in step c) comprises: c1) contacting the analyte bound to the magnetic particles with a detection molecule; and c2) obtaining a detection signal of the fluid in the reaction vessel. Eluting the detection molecules bound to the analyte.

  For any of the method embodiments described above, the magnetic field is generated by a magnetic assembly integrated in a heat block. The magnetic assembly integrated in the heat block may be any of the above, preferred, more preferred, or most preferred embodiments.

Figure 5 shows fluorescence images fired from 96-well plates with different amounts of magnetic particles. It shows the effect of different degrees of blocking of MGP. Figure 2 shows a schematic view of a preferred arrangement of magnets in a heating block. One possible arrangement of magnets and air gaps is shown. 1 shows an apparatus comprising a detection unit, a rack for holding a container, and a magnet coupled to the rack. FIG. 2 shows an apparatus with individual containers or containers that are part of a multi-well plate.

  FIG. 1 shows an image of fluorescence emitted from a 96 well microtiter plate (not all wells shown). Each well contains the same volume and concentration of fluorescent dye (50 nM of fluorescent dye is 50 μl and Tris buffer is 50 μl) and increased magnetic particles from left to right (Cobas Tacman ™ and Magna Pure) (Roche magnetic bead particles used for (trade name)). The first and last columns of the well contain a fluorescent dye without particles. It can be seen that the fluorescence intensity is highest when no magnetic particles are present and decreases with increasing amount of precipitated magnetic particles. From left to right, each well contains 0, 1, 2, 4, 6, 8, 12, 16 and 0 mg of magnetic particles, respectively.

  FIG. 2 shows the effect of different degrees of blocking of MGP. The fluorescence intensity in column 3 is significantly increased. Furthermore, FIG. 3 shows that increased strength can be achieved in the multiwell.

  FIG. 4 shows a schematic view of the reaction vessel (1) containing the liquid (4). The magnetic unit (3) is placed under the meniscus of the liquid (2) so that the magnetic particles (5) are sealed to the side walls of the reaction vessel. In order to minimize the loss of illuminating light collected by the detector placed above the well, the area of the aggregated beads projected onto the horizontal plane should be minimized. This can be achieved by adjusting the magnetic field so that the beads are skillfully concentrated just below the liquid surface. If the beads are pulled over the liquid surface, the beads tend to generate blisters that block some of the excitation and emission.

  FIG. 5 shows a schematic diagram of one possible arrangement of the air gap (6) and the magnetic unit (3), whereby the air gap is placed between the magnetic poles (black part, white part) of the magnetic unit. .

  FIG. 6 shows an apparatus with a container (1) that becomes each container or part of a multi-well plate. The container is held in a rack (7) and placed between the equipoles of the magnetic unit (3). The device also includes a detection unit (8) for detecting light emission (9). A lens (10) may be placed between the container and the detector.

Claims (17)

A rack for receiving at least one reaction vessel, the rack including at least one gap capable of receiving the reaction vessel;
One or more magnetic units for exerting a magnetic field on at least one reaction vessel containing magnetic particles capable of binding to an analyte contained in a fluid, wherein the one or more units comprise the rack The magnetic particles are sealed to the inner wall of the at least one reaction vessel by a magnetic field generated by the at least one magnetic unit.
A detection unit for detecting a signal of a fluid in the reaction vessel, wherein the detection unit is arranged in the apparatus for detecting a signal from the reaction vessel, The reaction vessel including the detection unit is held in the rack, and the detection unit detects a signal from the reaction vessel while holding the reaction vessel containing the magnetic particles in the rack. And an analysis processing and detection device provided in the device.   2. A device according to claim 1, characterized in that the rack is a thermally conductive rack, preferably a heat block.   3. A device according to claim 1 and / or 2, characterized in that the material of the rack consists of aluminum or silver.   4. The apparatus according to claim 1, wherein the magnetic field can be increased or decreased.   The one or more units for exerting a magnetic field on at least one reaction vessel containing magnetic particles in a fluid are one or more permanent magnets integrated in the rack. The apparatus of any one of 1-4.   6. The apparatus of claim 5, wherein the one or more permanent magnets are thermally stable at temperatures up to 140 ° C, preferably at temperatures up to 180 ° C, more preferably at temperatures up to 250 ° C. .   6. The apparatus of claim 5, wherein the one or more permanent magnets are made of an SmCo alloy.   8. The gap for receiving the reaction vessel of the rack is arranged between at least two of the permanent magnets so that the isomagnetic poles of the permanent magnet face the same gap. The apparatus of any one of these.   9. The apparatus according to claim 8, wherein the permanent magnet is placed under a liquid meniscus contained in a reaction vessel held by the rack.   The apparatus according to claim 1, wherein the detection unit is an optical detection unit. A method for quantifying the amount of analyte bound to magnetic particles present in a sample fluid in a reaction vessel, comprising:
The method
a) providing a biological sample containing the analyte in at least one reaction vessel;
b) binding magnetic particles to the analyte in the at least one reaction vessel;
c) processing the analyte bound to the magnetic particles in the at least one reaction vessel to detect the detection signal, which is a signal disturbed by the presence of the magnetic particles, wherein A processing step characterized in that the at least one reaction vessel is placed in a sample processing unit;
d) generating a suspended matter by using a magnetic field with respect to the contents of the at least one reaction vessel and sealing the magnetic particles on the inner wall of the reaction vessel;
e) a step of detecting a detection signal corresponding to the analyte concentration in the suspended matter in the reaction vessel in the presence of the magnetic particles, and the steps c) to e) are performed in the same analysis processing unit. A method characterized by that.   The method according to claim 11, wherein the specimen is a nucleic acid or a polypeptide.   13. A method according to claim 11 and / or 12, wherein the processing of the nucleic acid comprises amplification.   14. A method according to any one of claims 11 to 13, characterized in that the detection signal is an optical detection signal, preferably fluorescence.   15. The method according to any one of claims 11 to 14, wherein the treatment comprises elution of the base from magnetic beads into the fluid in the reaction vessel. The treatment of the polypeptide in step c)
c1) contacting the analyte bound to the magnetic particles with a detection molecule, preferably a labeled antibody capable of binding the analyte;
13. The method according to claim 12, further comprising the step of: c2) eluting the detection molecules bound to the specimen in order to obtain a detection signal in the fluid in the reaction vessel.   17. A method according to any one of claims 12 to 16, wherein the magnetic field is generated by a magnetic assembly integrated in a heat block.